In this revised competing application for renewal, we propose to build on key advances during the previous grant period, i.e., culture isolation of spotted fever group (SFG) tick symbionts; stable transformation of two SFG rickettsial tick symbionts to express GFP, and their use as model organisms to track routes of dispersal of SFG rickettsiae within ticks; identification of a plasmid in these rickettsiae having sequence homology with the R. felis plasmids, and discovery of plasmids in other rickettsial isolates, which is contrary to the paradigm that rickettsiae do not possess extrachromosomal DNA; and characterization of I. scapularis immune responses. We now propose to systematically probe rickettsial symbiont biology by genomic comparison, analysis of global gene expression, and development of improved transformation technology. To complement these efforts, we will test the ability of rickettsial transformants to colonize ticks, generating paratransgenic ticks. The goal of the proposed research is to develop genomic and transformation technologies that will enable us to critically evaluate the paratransgenesis paradigm as a strategy for analysis of the vector - microbe interaction. We propose to systematically probe rickettsial symbiont biology by genomic comparison, analysis of global gene expression, and development of improved transformation technology. Our central hypotheses are 1) A detailed comparison of symbiont and pathogen genomes and a functional analysis of key genes will a) reveal important details about the ability of symbionts to provide ticks with nutrients that are absent from the blood meal; b) define the potential of pathogens to be transmitted to vertebrate hosts and induce illness. 2) transgenic rickettsiae expressing reporter genes can be used to create a tick paratransgenesis model that will enable us to examine and identify the biological processes that lead to successful colonization of ticks by rickettsiae, a prerequisite for establishment of rickettsial transformants in ticks. There are 3 aims: 1) We will shotgun-sequence the Rickettsia peacockii symbiont genome and probe tiling arrays for global gene expression analysis and comparison with Rickettsia rickettsii in an effort to identify those genes and their products that make a symbiont versus those that make a pathogen. The results will provide a direct comparison of invasion and survival mechanisms used by symbiotic and pathogenic rickettsiae 2) We propose to develop novel transformation tools for SFG rickettsiae, i.e., a shuttle vector for transformation of rickettsiae based on the plasmid, pRM, that we have identified in Rickettsia monacensis. In addition, we will optimize the Himar1 transposase system that we have successfully applied to transformation of tick-borne Anaplasma phagocytophilum, for SFG rickettsiae. This will address the recently found bias of Tn5 for GC rich regions. 3) Finally, in aim 3, we propose to carry out real-time imaging of fluorescent protein expressing rickettsiae and analyze rickettsial movement in ticks to probe mechanisms of tick colonization, a prerequisite for establishment of both symbionts and pathogens.